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. Author manuscript; available in PMC: 2012 Sep 1.
Published in final edited form as: Clin Chem. 2011 Jul 18;57(9):1279–1285. doi: 10.1373/clinchem.2010.161174

Quantification of 1α,25 Dihydroxy Vitamin D by Immunoextraction and Liquid Chromatography-Tandem Mass Spectrometry

Frederick G Strathmann 1, Thomas J Laha 1, Andrew N Hoofnagle 1,2
PMCID: PMC3261234  NIHMSID: NIHMS346873  PMID: 21768219

Abstract

Background

1α,25-dihydroxy vitamin D [1,25(OH)2D] is the active metabolite of vitamin D. Antibody-based detection methods lack specificity, but when combined with isotope dilution-UPLC-tandem mass spectrometry, immunoextraction provides an attractive method for 1,25(OH)2D. We developed a method for simultaneous quantification of 1,25(OH)2D2 and 1,25(OH)2D3 with a 4.6 min instrument cycle time. Results are available 36 h after sample preparation begins.

Methods

Sample preparation consisted of protein precipitation, immunoextraction with solid-phase anti-1,25(OH)2D antibody, and derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione. Analytes were resolved using reversed-phase UPLC and quantified using positive ion electrospray ionization-tandem mass spectrometry. Hexadeuterated 1,25(OH)2D3 and 1,25(OH)2D2 were used as internal standards. Method comparisons were performed against the DiaSorin RIA and an LC-MS/MS method available at a reference laboratory.

Results

1,25(OH)2D3 intra-assay and inter-assay imprecision was 5.6% and 8.0% (120 pmol/L) and 8.7% and 13% (48 pmol/L). Limits of detection and quantification were 1.5 pmol/L and 3.0 pmol/L, respectively. 1,25(OH)2D2 intra-assay and inter-assay imprecision was 8.7% and 11% (186 pmol/L) and 11% and 13% (58 pmol/L). Limits of detection and quantification were 1.5 pmol/L. Comparison with RIA had a proportional bias of 0.75, constant bias of −4.1 and Pearson correlation (r2) of 0.31. Comparison with a reference LC-MS/MS assay had a porportional bias of 0.89, constant bias of 3.7 and Pearson correlation (r2) of 0.88.

Conclusion

Protein precipitation with antibody-based extraction is effective for sample preparation prior to LC-MS/MS analysis of derivatized 1,25(OH)2D. This method appears to have improved specificity over a clinically-used RIA with low imprecision and limits of detection.

Keywords: Liquid chromatography-tandem mass spectrometry, vitamin D, dihydroxy vitamin D, immunoaffinity, PTAD, protein precipitation

Introduction

The introduction of immunoassay-based detection methods to the clinical laboratory greatly improved our ability to diagnose and monitor disease. Unfortunately, even with the advent of new, more analytically sensitive detection methods, the inherent flaws of immunoassays remain (1, 2). Much like the dramatic effect immunoassays had on clinical testing, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has changed the face of laboratory testing by providing multiplexed, high-throughput analyses with better analytical specificity than that currently possible using “antibody-only” detection methods (3). Various LC-MS/MS-based methods for measuring 25-hydroxy vitamin D [25(OH)D] have been implemented in clinical laboratories across the country resulting in a substantial improvement in the accuracy of vitamin D measurement (4–9).

The metabolism of vitamin D into a biologically active form includes two separate hydroxylation steps. The first occurs in the liver and produces 25(OH)D. With a circulating concentration in the low nmol/L range and a long half-life, this 25-hydroxylated form serves as an indicator of vitamin D stores but has low affinity for the vitamin D receptor and consequently low biological activity. Transit to the kidneys bound by vitamin D binding protein is followed by a second hydroxylation reaction to 1α,25 dihydroxy vitamin D [1,25(OH)2D]. It is this dihydroxylated form that is responsible for the biological activities ascribed to vitamin D, including increased intestinal absorption of calcium and phosphate, bone mineralization, suppression of parathyroid hormone synthesis and secretion, and parathyroid cell proliferation (10, 11). The 1α-hydroxylase enzyme is also expressed locally in other cells, where 1,25(OH)2D likely has autocrine or paracrine effects (12, 13). While it is well established that the measurement of 25(OH)D is useful in determining vitamin D stores, analysis of this vitamin D metabolite provides no information about the active dihydroxylated metabolite of vitamin D. On the other hand, the measurement of plasma concentrations of the biologically active hormone, 1,25(OH)2D, is important for the diagnosis and management of patients with chronic kidney disease (14), oncogenic osteomalacia syndrome (15) and acquired or inborn errors of phosphate homeostasis (16).

One challenge associated with measuring 1,25(OH)2D is the 1,000-fold lower concentration in plasma compared with the more widely measured precursor 25(OH)D (4, 17–19). Compounding the problem of low concentration is the poor ionization of the analyte and the potential difficulties introduced by the derivatization necessary for adequate analytical sensitivity (20, 21). Additionally, inferior sample preparation techniques can have a substantial negative impact on the performance of clinical LC-MS/MS-based methods (22).

Different acceptability criteria exist between methods used for research and methods used for routine clinical diagnosis. Several methods have been described for the measurement of 1,25(OH)2D by LC-MS/MS, each with their own inherent advantages and disadvantages (4, 20, 23, 24). We describe here an immunoaffinity UPLC-MS/MS method with clinically relevant limits of detection and imprecision when compared with current methodologies.

Materials and Methods

Chemicals and human samples

All chemicals were purchased from Fisher Scientific unless otherwise noted. The use of human specimens was approved by the Human Subjects Division at the University of Washington.

Assay time and cost

A single batch included six calibrators, one high control, one low control, and up to 21 samples. Total assay time was approximately 7.5 h, split between protein precipitation (1.5 h per batch), immunoextraction (5 h per batch), derivatization and resuspension (1 h per batch) and analysis (4.7 min per injection). Cost analysis was conducted using 7.5 h per batch (0.28 h per sample), reagents and supplies of $351.54 per batch ($16.74 per sample) and instrument time of 2.1 h per batch (4.7 min per injection).

Protein precipitation

The protein precipitation solution consisted of 8 µL of a 24 nmol/L mixture of hexadeuterated 1,25(OH)2D3 and 1,25(OH)2D2 (Medical Isotopes, Inc.) and 992 µL of 80:20% methanol:acetonitrile, which was added to 400 µL of sample in 1.7mL polypropylene microcentrifuge tubes, inverted 3 times and vortexed for 5 min. After centrifugation at 17,100×g for 10 min, the resulting supernatant was evaporated at 45°C under forced air and stored overnight at −20°C.

Immunoextraction

Dried samples were resuspended in a solution containing 500 µL of solid phase monoclonal anti-1,25(OH)2D (IDS, England) for 2 hours with end-over-end rotation in the same polypropylene microcentrifuge tubes. After 4 consecutive washes with 1 mL of water, bound 1,25(OH)2D was eluted using 500 µL of 100% ethanol and the solid phase antibody was discarded. Samples were dried by vacuum centrifugation for 2 h to ensure complete removal of ethanol and residual water.

Derivitization

Samples were reconstituted in 13 µL of a 9 mmol/L 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD; Sigma-Aldrich, St. Louis) prepared in 100% acetonitrile, vortexed briefly and centrifuged for 1 min at 17,100×g. After incubating for 30 min at room temperature, samples were centrifuged for 15 seconds at 17,000×g and the resuspended samples (~13 µL) were transferred to polypropylene autosampler vials containing 17 µL of HPLC grade water to quench the non-reacted PTAD and to provide a sample composition matching the initial chromatographic conditions. For each sample, the mean response of two 10 µL injections was used for further analysis.

Chromatography and mass spectrometry

Analytes in the 10 µL injection volume were resolved by reversed phase liquid chromatography using a Waters Acquity UPLC with a BEH-C18 (2.1 mm × 50 mm × 1.7 µm) column, a 50 µL sample loop and the inlet method detailed in Supplemental Table 1. We quantified the analytes by using isotope dilution-multiple reaction monitoring on a Waters Xevo mass spectrometer (transitions: 1,25(OH)2D3, 574.37 and 592.37 > 314.12; 1,25(OH)2D2, 586.37 > 314.13; 1,25(OH)2D3-d6, 580.37 > 314.13; 1,25(OH)2D2-d6, 592.37 > 314.12). Two transitions were monitored for 1,25(OH)2D3 due to an inconsistent water loss at the source that would otherwise have contributed to increased imprecision of the assay (see Results for further explanation). Instrument specific cone and collision parameters were derived experimentally. Total instrument analysis time was 4.6 min per injection.

Calibration and quality control

Calibrators containing both 1,25(OH)2D3 and 1,25(OH)2D2 at 12, 24, 120, 240 and 480 pmol/L were prepared in 4% bovine serum albumin (BSA) and frozen at −20°C. Calibrator concentration was confirmed by measuring the absorbance of the prepared stock solutions (23.3 µmol/L for 1,25(OH)2D2 and 24.4 µmol/L of 1,25(OH)2D3) in ethanol using a Beckman 7000 series spectrophotometer set to 265nm and extinction coefficients of 18900 and 18300 for 1,25(OH)2D3 and 1,25(OH)2D2, respectively. Analyte concentrations were determined using a calibration curve based on the response ratio (peak area of unlabeled vitamin divided by peak area of labeled internal standard). For quality control materials, 50 human serum samples were pooled, aliquoted and frozen at −20°C. Low concentration control material was also made by diluting the starting pool 1:4 with 4% BSA.

Evaluation of assay performance

Intraassay imprecision was evaluated by running 20 replicates of both high and low controls in a single run. Interassay imprecision was estimated over a 3 week period using four separate runs with 5 replicates of low and high controls in each run. Analtyical recovery was determined by spiking high controls with one of two concentrations of internal standards before the protein precipitation (Pre-crash), after the protein precipitation but before the immunoextraction (Pre-IE) or after the immunoextraction (Post-IE). Analytical recoveries were calculated for each sample (run in triplicate) by dividing the peak areas of the internal standards for the Pre-crash or Pre-IE samples by the Post-IE peak areas and multiplying by 100 to obtain a percent recovery. Clinical recoveries were determined by spiking high controls with one of two concentrations of 1,25(OH)2D3 and 1,25(OH)2D2. Clinical recoveries were calculated by dividing the measured concentration (after subtraction of the predetermined levels of 1,25(OH)2D2 and 1,25(OH)2D3 already present in the sample) by the expected concentration. Interference from lipemia and total protein was determined in a similar manner to that described for the clinical recoveries using samples with increased total protein, direct LDL and/or triglycerides. Ion suppression was assessed by postcolumn infusion of deuterated internal standards with injection of an immunoextracted serum sample (22). A reference range study was performed using 40 independent samples from healthy, blood-bank donors with values selected by non-parametric analysis (central 95%).

Determination of sample type acceptability

Preanalytical effects were determined by comparing values obtained from fresh serum, lithium heparin and potassium EDTA plasma samples to samples stored for 24 h at 4°C or −20°C. A one sample t-test was used to determine if a statistically significant bias existed for all anticoagulants and storage conditions compared to frozen serum.

Data analysis

Data was tabulated in a Microsoft Excel spreadsheet, with statistical analyses and graphing performed with the R statistics language (http://www.r-project.org/) and ggplot2 graphics package (http://cran.r-project.org/web/packages/ggplot2/index.html). Deming regression from the R package MethComp (http://cran.r-project.org/web/packages/MethComp/) was conducted using 1000 boot-strap iterations and variance ratio [LC-MS/MS:RIA = 0.38](25).

Results

Assay performance

Method validation results are presented in Tables 1 and 2 for 1,25(OH)2D2 and 1,25(OH)2D3. The combination of methanol:acetonitrile protein precipitation and immunoextraction yielded intraassay and interassay CVs less than 14% (Table 1), consistent with published imprecision of the DiaSorin Assay (18). The limit of detection (defined as a signal to noise ratio greater than 8) was 1.5 pmol/L for both 1,25(OH)2D3 and 1,25(OH)2D2. Limits of quantification (defined as the lowest concentration tested with a CV of less than 20%) were 3.0 pmol/L [1,25(OH)2D3] and 1.5 pmol/L [1,25(OH)2D2]. Analytical recoveries were equal to or greater than 40% with clinical recoveries ranging from 91% to 120% (Table 2). No interference was seen from hyperproteinemia (mean total protein 87 g/L) or hyperlipidemia (mean triglycerides > 6 mmol/L or LDL > 2.3 g/L). The analytical measurement range (AMR) for the assay was set at 12 pmol/L to 480 pmol/L for total 1,25(OH)2D to allow continued use of established calibrators and AMR validation during each run. Mixed results were produced from comparisons made between multiple storage conditions or anticoagulants (Supplemental Table 2). No statistically significant difference was found between fresh, frozen (−20°C) or refrigerated (4°C) serum or lithium-heparin plasma when stored for 24 hours; however, there was a significant decrease in concentration with EDTA-plasma regardless of storage conditions. In addition, the results of frozen samples tended to be higher than unfrozen samples, suggesting that freeze-thawing helps denature the binding proteins and aids extraction efficiency. Taken together, the observed effects, though not universally statistically significant, prompted us to make frozen serum the preferred specimen type and frozen lithium heparin anticoagulated plasma an acceptable alternative.

Table 1.

Intraassay and interassay imprecision.

Intraassay Imprecision
(n=20)		 Interassay Imprecision
(n=20)
Mean (pmol/L)a CV Mean (pmol/L)a CV
1,25(OH)2D3 125 5.6% 125 8.0%
39 8.7% 43 13%
1,25(OH)2D2 195 8.7% 182 11%
60 11% 58 13%
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a

To convert from pmol/L to pg/mL, divide 1,25(OH)2D3 by 2.4 and 1,25(OH)2D2 by 2.33.

Table 2.

Analytical and clinical recovery.

Concentration
(pmol/L)		 Clinical
Recovery		 Analytical
Recovery
(Pre-Crash)		 Analytical
Recovery
(Pre-IE)
1,25(OH)2D3 240 119% 40% 59%
48 91% 50% 60%
1,25(OH)2D2 117 112% 78% 116%
23 110% 82% 83%
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Development of a chromatographic method

A representative chromatogram from a patient sample for 1,25(OH)2D3, 1,25(OH)2D2 and the deuterated internal standards is shown in Fig. 1. The resolution between 1,25(OH)2D3 and 1,25(OH)2D2 was calculated to be 2.36, with retention factors of 5.21 (k'D3), 5.63 (k'D2) and a selectivity factor of 1.08. A combination of initial mobile phase conditions of 55/45 (buffer A to buffer B) and elution of 1,25(OH)2D3 and 1,25(OH)2D2 with a 40/60 composition was ideal for separation of analytes from residual matrix components, themselves eluting at a 5/95 buffer composition and capable of causing notable ion suppression (Figure 1, inset).

Figure 1. Representative chromatograms of 1,25(OH)2D3, 1,25(OH)2D2 and a qualitative ion suppression analysis.

Figure 1

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Traces of relative ion intensity from sera with 120 pmol/L of 1,25(OH)2D3 and 1,25(OH)2D2 plus addition of 200 pmol/L of internal standards. The inset is a tracing of ion suppression. The thin black line indicates the relative intensity of an infusion of internal standards as described in Materials and Methods. Representative peaks for 1,25(OH)2D3 and 1,25(OH)2D2 are filled in black and labeled as 1 and 2, respectively.

Variable fragmentation at the source

The resolution of the chromatographic method allowed us to identify a minor species of 1,25(OH)2D3 being detected in the 1,25(OH)2D2-d6 internal standard transition (Supplemental Figure 1), corresponding to 1,25(OH)2D3 without a water loss before the first quadrupole. In the current method, the majority of 1,25(OH)2D3 loses a hydrogen and the 25-hydroxyl group in the source, resulting in a dominant precursor ion of m/z 574.42, while a small fraction retains this water group and has m/z coinciding with the precursor ion of 1,25(OH)2D2-d6 at m/z 592.482. This minor ion of 1,25(OH)2D3 was found to be proportional to the concentration of total 1,25(OH)2D3, as illustrated by the chromatograms of the 1,25(OH)2D2-d6 transition in the standard curve shown in Supplemental Figure 1. However, the proportion of the minor ion was not consistent between injections of the same sample nor across samples with similar 1,25(OH)2D3 concentrations (varying from 15 to 35% of the total) and was present at all instrument parameter configurations tested. The inclusion of this minor ion in the total peak area for 1,25(OH)2D3 resulted in a substantial decrease in the imprecision of the assay for 1,25(OH)2D3 (8.7% vs. 11% at 48 pmol/L) and it was decided that continued monitoring and summation of both ion peak areas was necessary. A similar minor ion for 1,25(OH)2D2 was determined to be consistently present at less than 5% of the major peak and was therefore excluded from analysis.

Determination of immunoextraction specificity

Although detection by mass spectrometry is highly specific, we decided to more completely analyze immunoextracted samples by including transitions for known vitamin D metabolites to test for antibody cross-reactivity. In addition to the expected 1,25(OH)2D species, we also identified 24,25(OH)2D3 and 25(OH)D3 (Fig. 2). These identified metabolites were well-resolved chromatographically from 1,25(OH)2D species and did not interfere with the transitions monitored for the quantification of 1,25(OH)2D.

Figure 2. Vitamin D metabolites immunoextracted from a human sample by the anti-1,25(OH)2D antibody.

Figure 2

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Chromatograms from an analysis of immunoextracted patient sera modified to include transitions corresponding to 24,25(OH)2D3 (574.34 > 298.12) and 25(OH)D3 (558.34 > 298.12). 24,25(OH)2D3 and 25(OH)D3 are well separated from both 1,25(OH)2D3 and 1,25(OH)2D2. The internal standard is shown for 1,25(OH)2D2 due to an undetectable amount of endogenous analyte in this sample.

Method comparisons

Method comparisons were performed with patient results obtained from an outside laboratory using the DiaSorin RIA assay and a reference laboratory LC-MS/MS assay (Fig. 3). When compared to RIA, the LC-MS/MS method had a proportional bias of 0.75, a constant bias of −4.1 and a Pearson correlation coefficient (r2) of 0.31. Use of a Bland-Altman plot demonstrated a statistically significant negative bias (r = −0.41, p < 0.001) between the LC-MS/MS and RIA methods. These results support the proposal that LC-MS/MS detection of analytes is often more specific than detection by competitive immunoassay, which are known to be suffer from cross-reactivity (Fig. 2), and provide a plausible explanation for the lack of agreement between methods. However, the presented data cannot rule out differences in calibration between LC-MS/MS and RIA as an alternate explanation for the observed bias. When compared to a reference LC-MS/MS method, the presented method for total 1,25(OH)2D had a porportional bias of 0.89, constant bias of 3.7 and Pearson correlation (r2) of 0.88 (Figure 3). For 1,25(OH)D3, there was a proportional bias of 0.99, constant bias of 0.83 and Pearson correlation (r2) of 0.95.

Figure 3. Method comparison of the new LC-MS/MS assay with a 1,25(OH)2D radioimmunoassay and reference LC-MS/MS method.

Figure 3

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(Left) Total 1,25(OH)2D (pmol/L) measured with the DiaSorin RIA compared to total 1,25(OH)2D measured by LC-MS/MS (n=116). The black dotted line is the line of identity and the solid black line is the Deming regression line (−4.1 + 0.76×; r2 (Pearson) = 0.31). (Right) Comparison with a reference LC-MS/MS assay had a porportional bias of 0.89, constant bias of 3.7 and Pearson correlation (r2) of 0.88.

(Bottom) Bland-Altman plots of the average concentration against the relative difference for RIA versus LC-MS/MS and LC-MS/MS versus reference LC-MS/MS results. The mean difference with ± 2 SD of relative differences is shown (dashed lines).

Reference interval study

We conducted a reference interval study using 40 specimens from healthy, blood bank donors. Specimens were analyzed over multiple days and nonparametric analysis was used to determine the central 95% of the data. The reference interval established for total 1,25(OH)2D was 48 to 168 pmol/L (19 to 67 pg/mL).

Discussion

Over the past several years, serum measurements of 25(OH)D have rapidly increased in testing volume. The widespread implementation of LC-MS/MS-based measurements has allowed for increased specificity and cost savings when compared with immunoassay based methods. Although 25(OH)D is the major circulating metabolite of vitamin D and the form most often used clinically, it is the active 1,25-dihydroxylated form of the hormone that is responsible for its biological effects [e.g., intestinal calcium uptake, bone resorption and parathyroid hormone suppression (13, 26, 27)]. The clinical utility of measuring 1,25(OH)2D is not fully understood in the general population, but it is clear that associations are being made between this active metabolite of vitamin D and disease (28–33). Further, the use of LC-MS/MS to measure 1,25(OH)2D has been challenging due low circulating concentrations, emphasized by a reported reference interval of approximately 36 to 144 pmol/L or 15 to 60 pg/mL (34), with variations existing based upon testing methodology.

Previously published LC-MS/MS methods for the measurement of 1,25(OH)2D represent significant strides in our ability to accurately quantify vitamin D metabolism. To detect the low endogenous concentrations of 1,25(OH)2D, excellent limits of detection are needed while maintaining the highest possible specificity. To achieve these necessary performance characteristics, we first focused on sample preparation. Our decision to use immunoextraction as a sample preparation method was based on previously reported success with other small molecules in a wide range of complex matrices (35–40). We also carefully evaluated different chromatographic approaches in order to separate 1,25(OH)2D from related metabolites, species with and without water loss in the source, and matrix components. Derivatization of the resultant 1,25(OH)2D was also critical and consistent with previous reports (5, 6, 20, 21). The presented combination of immunoextraction and optimized chromatography yielded a robust, clinically validated method with an instrument cycle time of less than 5 minutes. This method represents a significant improvement over "antibody-only" detection methods and adds to the concern regarding accuracy and reproducibility with immunoassay measurements of analytes at low concentrations.

Due to the poor comparison with the RIA method, we chose a reference LC-MS/MS method for further comparison. It is unclear why the two methods differ with respect to 1,25(OH)2D2. It is possible that difference may lie in the variable fragementation in the soure we observed for 1,25(OH)2D3. Our chromatographic separation provides the necessary resolution between 1,25(OH)3D2 and 1,25(OH)2D3 to eliminate cross-reactivty of the 1,25(OH)2D3 into the 1,25(OH)2D2-d6 internal standard transition. Less efficient resolution of 1,25(OH)2D3 and 1,25(OH)2D2 combined with the variability of the water loss at the source for 1,25(OH)2D3 can interfere with nonresolved 1,25(OH)2D2-d6 and compromise the quantification of 1,25(OH)2D2.

The existence of a wide range of antibodies currently used in clinical immunoassays could allow the development of cost-effective mechanisms for transitioning current immunodetection methods to combined immunoextraction LC-MS/MS methods.

Supplementary Material

Supplemental Material

Acknowledgments

This work was funded by the University of Washington (UW) Clinical Mass Spectrometry Facility and the UW Nutrition and Obesity Research Center (NIH P30DK035816). The authors thank Dr. Ravinder Singh, Brandon Hill and Mayo Medical Laboratories for providing LC-MS/MS measurements.

Abbreviations

LC-MS/MS

Liquid chromatography-tandem mass spectrometry

PTAD

4-phenyl-1,2,4-triazoline-3,5-dione

References

  • 1.Hoofnagle A, Wener M. The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry. Journal of immunological methods. 2009;347:3–11. doi: 10.1016/j.jim.2009.06.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Chace D. Mass spectrometry in the clinical laboratory. Chemical reviews. 2001;101:445–477. doi: 10.1021/cr990077+. [DOI] [PubMed] [Google Scholar]
  • 3.Vogeser M, Parhofer KG. Liquid chromatography tandem-mass spectrometry (LC-MS/MS)--technique and applications in endocrinology. Exp Clin Endocrinol Diabetes. 2007;115:70. doi: 10.1055/s-2007-981458. [DOI] [PubMed] [Google Scholar]
  • 4.Duan X, Weinstock-Guttman B, Wang H, Bang E, Li J, Ramanathan M, Qu J. Ultrasensitive quantification of serum vitamin D metabolites using selective solid-phase extraction coupled to microflow liquid chromatography and isotope-dilution mass spectrometry. Analytical chemistry. 2010;82:2488–2497. doi: 10.1021/ac902869y. [DOI] [PubMed] [Google Scholar]
  • 5.Eyles D, Anderson C, Ko P, Jones A, Thomas A, Burne T, et al. A sensitive LC/MS/MS assay of 25OH vitamin D3 and 25OH vitamin D2 in dried blood spots. Clinica chimica acta; international journal of clinical chemistry. 2009;403:145–151. doi: 10.1016/j.cca.2009.02.005. [DOI] [PubMed] [Google Scholar]
  • 6.Higashi T, Shibayama Y, Fuji M, Shimada K. Liquid chromatography-tandem mass spectrometric method for the determination of salivary 25-hydroxyvitamin D3: a noninvasive tool for the assessment of vitamin D status. Analytical and bioanalytical chemistry. 2008;391:229–238. doi: 10.1007/s00216-007-1780-3. [DOI] [PubMed] [Google Scholar]
  • 7.Singh RJ, Taylor RL, Reddy GS, Grebe SKG. C-3 epimers can account for a significant proportion of total circulating 25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. The Journal of clinical endocrinology and metabolism. 2006;91:3055–3061. doi: 10.1210/jc.2006-0710. [DOI] [PubMed] [Google Scholar]
  • 8.Saenger AK, Laha TJ, Bremner DE, Sadrzadeh SMH. Quantification of serum 25-hydroxyvitamin D(2) and D(3) using HPLC-tandem mass spectrometry and examination of reference intervals for diagnosis of vitamin D deficiency. American journal of clinical pathology. 2006;125:914–920. doi: 10.1309/J32U-F7GT-QPWN-25AP. [DOI] [PubMed] [Google Scholar]
  • 9.Phinney KW. Development of a standard reference material for vitamin D in serum. The American journal of clinical nutrition. 2008;88:511S–512S. doi: 10.1093/ajcn/88.2.511S. [DOI] [PubMed] [Google Scholar]
  • 10.DeLuca HF, Plum LA, Clagett-Dame M. Selective analogs of 1alpha,25-dihydroxyvitamin D3 for the study of specific functions of Vitamin D. J Steroid Biochem Mol Biol. 2007;103:263–268. doi: 10.1016/j.jsbmb.2006.12.005. [DOI] [PubMed] [Google Scholar]
  • 11.Fleet JC, Bradley J, Reddy GS, Ray R, Wood RJ. 1 alpha,25-(OH)2-vitamin D3 analogs with minimal in vivo calcemic activity can stimulate significant transepithelial calcium transport and mRNA expression in vitro. Archives of biochemistry and biophysics. 1996;329:228–234. doi: 10.1006/abbi.1996.0213. [DOI] [PubMed] [Google Scholar]
  • 12.Prosser DE, Jones G. Enzymes involved in the activation and inactivation of vitamin D. Trends Biochem Sci. 2004;29:73. doi: 10.1016/j.tibs.2004.10.005. [DOI] [PubMed] [Google Scholar]
  • 13.Gallieni M, Cozzolino M, Fallabrino G, Pasho S, Olivi L, Brancaccio D. Vitamin D: physiology and pathophysiology. The International journal of artificial organs. 2009;32:87–94. doi: 10.1177/039139880903200205. [DOI] [PubMed] [Google Scholar]
  • 14.Williams S, Malatesta K, Norris K. Vitamin D and chronic kidney disease. Ethn Dis. 2009;19 S5-8-11. [PMC free article] [PubMed] [Google Scholar]
  • 15.Hannan FM, Athanasou NA, Teh J, Gibbons CL, Shine B, Thakker RV. Oncogenic hypophosphataemic osteomalacia: biomarker roles of fibroblast growth factor 23, 1,25-dihydroxyvitamin D3 and lymphatic vessel endothelial hyaluronan receptor 1. Eur J Endocrinol. 2008;158:265–271. doi: 10.1530/EJE-07-0485. [DOI] [PubMed] [Google Scholar]
  • 16.Bergwitz C, Juppner H. Disorders of phosphate homeostasis and tissue mineralisation. Endocr Dev. 2009;16:133–156. doi: 10.1159/000223693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Hollis BW. Assay of circulating 1,25-dihydroxyvitamin D involving a novel single-cartridge extraction and purification procedure. Clin Chem. 1986;32:2060–2063. [PubMed] [Google Scholar]
  • 18.Clive DR, Sudhaker D, Giacherio D, Gupta M, Schreiber MJ, Sackrison JL, MacFarlane GD. Analytical and clinical validation of a radioimmunoassay for the measurement of 1,25 dihydroxy vitamin D. Clin Biochem. 2002;35:517–521. doi: 10.1016/s0009-9120(02)00378-8. [DOI] [PubMed] [Google Scholar]
  • 19.Seiden-Long I, Vieth R. Evaluation of a 1,25-dihydroxyvitamin D enzyme immunoassay. Clin Chem. 2007;53:1104–1108. doi: 10.1373/clinchem.2006.077560. [DOI] [PubMed] [Google Scholar]
  • 20.Aronov PA, Hall LM, Dettmer K, Stephensen CB, Hammock BD. Metabolic profiling of major vitamin D metabolites using Diels-Alder derivatization and ultra-performance liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem. 2008;391(5):1917–1930. doi: 10.1007/s00216-008-2095-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Weiskopf AS, Vouros P, Cunniff J, Binderup E, Björkling F, Binderup L, et al. Examination of structurally selective derivatization of vitamin D(3) analogues by electrospray mass spectrometry. J Mass Spectrom. 2001;36:71–78. doi: 10.1002/jms.105. [DOI] [PubMed] [Google Scholar]
  • 22.Annesley TM. Ion suppression in mass spectrometry. Clin Chem. 2003;49:1041–1044. doi: 10.1373/49.7.1041. [DOI] [PubMed] [Google Scholar]
  • 23.Kissmeyer AM, Sonne K. Sensitive analysis of 1alpha,25-dihydroxyvitamin D3 in biological fluids by liquid chromatography-tandem mass spectrometry. J Chromatogr A. 2001;935:103. doi: 10.1016/s0021-9673(01)00985-2. [DOI] [PubMed] [Google Scholar]
  • 24.Vreeken RJ, Honing M, van Baar BL, Ghijsen RT, de Jong GJ, Brinkman UA. On-line post-column Diels-Alder derivatization for the determination of vitamin D3 and its metabolites by liquid chromatography/thermospray mass spectrometry. Biol Mass Spectrom. 1993;22:621–632. doi: 10.1002/bms.1200221102. [DOI] [PubMed] [Google Scholar]
  • 25.Linnet K. Performance of Deming regression analysis in case of misspecified analytical error ratio in method comparison studies. Clin Chem. 1998;44:1024–1031. [PubMed] [Google Scholar]
  • 26.DeLuca HF. Evolution of our understanding of vitamin D. Nutr Rev. 2008;66:S73–S87. doi: 10.1111/j.1753-4887.2008.00105.x. [DOI] [PubMed] [Google Scholar]
  • 27.Haarburger D, Hoffman M, Erasmus RT, Pillay TS. Relationship between vitamin D, calcium and parathyroid hormone in Cape Town. J Clin Pathol. 2009;62:567–569. doi: 10.1136/jcp.2008.062877. [DOI] [PubMed] [Google Scholar]
  • 28.Tuohimaa P, Keisala T, Minasyan A, Cachat J, Kalueff A. Vitamin D, nervous system and aging. Psychoneuroendocrinology. 2009;34 Suppl 1:S278–S286. doi: 10.1016/j.psyneuen.2009.07.003. [DOI] [PubMed] [Google Scholar]
  • 29.Annweiler C, Schott A-M, Berrut G, Chauviré V, Le Gall D, Inzitari M, Beauchet O. Vitamin D and ageing: neurological issues. Neuropsychobiology. 2010;62:139–150. doi: 10.1159/000318570. [DOI] [PubMed] [Google Scholar]
  • 30.Masoumi A, Goldenson B, Ghirmai S, Avagyan H, Zaghi J, Abel K, et al. 1alpha,25-dihydroxyvitamin D3 interacts with curcuminoids to stimulate amyloid-beta clearance by macrophages of Alzheimer's disease patients. J Alzheimers Dis. 2009;17:703–717. doi: 10.3233/JAD-2009-1080. [DOI] [PubMed] [Google Scholar]
  • 31.Driver JP, Foreman O, Mathieu C, van Etten E, Serreze DV. Comparative therapeutic effects of orally administered 1,25-dihydroxyvitamin D(3) and 1alpha-hydroxyvitamin D(3) on type-1 diabetes in non-obese diabetic mice fed a normal-calcaemic diet. Clin Exp Immunol. 2008;151:76–85. doi: 10.1111/j.1365-2249.2007.03537.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Marniemi J, Alanen E, Impivaara O, Seppanen R, Hakala P, Rajala T, Ronnemaa T. Dietary and serum vitamins and minerals as predictors of myocardial infarction and stroke in elderly subjects. Nutr Metab Cardiovasc Dis. 2005;15:188–197. doi: 10.1016/j.numecd.2005.01.001. [DOI] [PubMed] [Google Scholar]
  • 33.Meehan TF, DeLuca HF. The vitamin D receptor is necessary for 1alpha,25-dihydroxyvitamin D(3) to suppress experimental autoimmune encephalomyelitis in mice. Arch Biochem Biophys. 2002;408:200–204. doi: 10.1016/s0003-9861(02)00580-5. [DOI] [PubMed] [Google Scholar]
  • 34.Tietz NW, Burtis CA, Ashwood ER, Bruns DE. Tietz textbook of clinical chemistry and molecular diagnostics. 4th ed. St. Louis, Mo: Elsevier Saunders; 2006. :xxxvi, 2412 p., [8] p. of platespp. [Google Scholar]
  • 35.Cai J, Henion J. Quantitative multi-residue determination of beta-agonists in bovine urine using on-line immunoaffinity extraction-coupled column packed capillary liquid chromatography-tandem mass spectrometry. Journal of chromatography B, Biomedical sciences and applications. 1997;691:357–370. doi: 10.1016/s0378-4347(96)00433-1. [DOI] [PubMed] [Google Scholar]
  • 36.Cai J, Henion J. On-line immunoaffinity extraction-coupled column capillary liquid chromatography/tandem mass spectrometry: trace analysis of LSD analogs and metabolites in human urine. Analytical chemistry. 1996;68:72–78. doi: 10.1021/ac950763i. [DOI] [PubMed] [Google Scholar]
  • 37.Nedved ML, Habibi-Goudarzi S, Ganem B, Henion JD. Characterization of benzodiazepine "combinatorial" chemical libraries by on-line immunoaffinity extraction, coupled column HPLC-ion spray mass spectrometry-tandem mass spectrometry. Analytical chemistry. 1996;68:4228–4236. doi: 10.1021/ac9603035. [DOI] [PubMed] [Google Scholar]
  • 38.Onorato J, Henion JD. Evaluation of triterpene glycoside estrogenic activity using LC/MS and immunoaffinity extraction. Analytical chemistry. 2001;73:4704–4710. doi: 10.1021/ac010409m. [DOI] [PubMed] [Google Scholar]
  • 39.Rule GS, Henion JD. Determination of drugs from urine by on-line immunoaffinity chromatography-high-performance liquid chromatography-mass spectrometry. Journal of chromatography. 1992;582:103–112. doi: 10.1016/0378-4347(92)80308-d. [DOI] [PubMed] [Google Scholar]
  • 40.Rule GS, Mordehai AV, Henion J. Determination of carbofuran by on-line immunoaffinity chromatography with coupled-column liquid chromatography/mass spectrometry. Analytical chemistry. 1994;66:230–235. doi: 10.1021/ac00074a009. [DOI] [PubMed] [Google Scholar]

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